Understanding how new species form is a fundamental problem in biology. Speciation occurs as reproductive barriers form between diverging populations. One type of reproductive barrier is the sterility and lethality of interspecific hybrids. These hybrid incompatibilities are caused by deleterious interactions between genes that have evolved independently in each diverging population. This proposal is based on the previous identification of two genes, Hmr and Lhr, which interact to cause hybrid lethality between the fruit fly species Drosophila melanogaster and Drosophila simulans. A major goal is to understand how these genes kill hybrids, and how the interaction between them is related to their patterns of DNA sequence divergence. One specific hypothesis is that Hmr and Lhr cause hybrid lethality by disrupting the structure and regulation of chromatin, the complex of DNA and associated proteins that controls and is essential for gene regulation, chromosome replication and cell division, and the maintenance of nuclear morphology. High-resolution imaging of chromatin-associated proteins and cytological studies of interphase nuclei will be used to test this hypothesis. The Hmr and Lhr genes encode proteins that have relatively high sequence divergence between the hybridizing species. A second hypothesis is that these diverged proteins will localize to different DNA sequences through their interactions with chromatin. This hypothesis will be tested by cytological methods and by performing chromatin immunoprecipitation (ChIP) followed by short- read sequencing of the recovered DNA. Interactions between the proteins encoded by Hmr and Lhr will also be investigated to determine whether these interactions are disrupted in interspecific hybrids. One striking aspect of the divergence of the Lhr gene is that D. simulans contains a substantial insertion in its coding region compared to D. melanogaster. An unusual Lhr allele has been discovered from D. simulans that lacks this insertion and has additional sequence changes compared to wild type alleles. This allele does not cause hybrid lethality and it will be investigated in order to determine whether the insertion contributes to functional differences in the Lhr gene between D. melanogaster and D. simulans. A region of the genome has been found that appears to contain a new major-effect hybrid lethality gene. This gene will be identified in order to determine whether it interacts with Hmr and Lhr or instead defines an independent pathway leading to hybrid lethality. PUBLIC HEALTH RELEVANCE: This project will provide a mechanistic understanding of how barriers between species form. The genes that form these barriers are part of what makes each species unique. Understanding the consequences of the evolutionary patterns of these genes will help to reveal how species including humans maintain a functionally coherent genome.